The present invention relates to the field of polymer screening. More specifically, in one embodiment the invention provides an improved polymer library and method of using the library to identify a polymer sequence that is complementary to a receptor.
Many assays are available for measuring the binding affinity of receptors and ligands, but the information which can be gained from such experiments is often limited by the number and type of ligands which are available. Small peptides are an exemplary system for exploring the relationship between structure and function in biology. When the twenty naturally occurring amino acids are condensed into peptides they form a wide variety of three-dimensional configurations, each resulting from a particular amino acid sequence and solvent condition. The number of possible pentapeptides of the 20 naturally occurring amino acids, for example, is 205 or 3.2 million different peptides. The likelihood that molecules of this size might be useful in receptor-binding studies is supported by epitope analysis studies showing that some antibodies recognize sequences as short as a few amino acids with high specificity.
Prior methods of preparing large numbers of different oligomers have been painstakingly slow when used at a scale sufficient to permit effective rational or random screening. For example, the “Merrifield” method, described in Atherton et al., “Solid Phase Peptide Synthesis,” IRL Press, (1989), incorporated herein by reference for all purposes, has been used to synthesize peptides on a solid support such as pins or rods. The peptides are then screened to determine if they are complementary to a receptor. Using the Merrifield method, it is not economically practical to screen more than a few peptides in a day.
Similar problems are encountered in the screening of other polymers having a diverse basis set of monomers. For example, various methods of oligonucleotide synthesis such as the phosphate-triester method and the phosphotriester method, described in Gait, “Oligonucleotide Synthesis,” IRL Press, (1990), incorporated herein by reference for all purposes, have similar limitations when it is desired to synthesize many diverse oligonucleotides for screening.
To screen a larger number of polymer sequences more advanced techniques have been disclosed. For example, Pirrung et al., WO 90/15070 U.S. Pat. No. 5,143,854, incorporated herein by reference for all purposes, describes a method of synthesizing a large number of polymer sequences on a solid substrate using light directed methods. Dower et al., U.S. application Ser. No. 07/762,522, also incorporated by reference herein for all purposes, describes a method of synthesizing a library of polymers and a method of use thereof. The polymers are synthesized on beads, for example. A first monomer is attached to a pool of beads. Thereafter, the pool of beads is divided, and a second monomer is attached. The process is repeated until a desired, diverse set of polymers is synthesized.
Other methods of synthesizing and screening polymers have also been proposed. For example, Houghten et al., “Generation and Use of Synthetic Peptide Combinatorial Libraries for Basic Research and Drug Discovery,” Nature (1991) 354:84-86, discuss a method of generating peptide libraries that are used for screening the peptides for biological activity (see also, Houghton et al., “The Use of Synthetic Peptide Combinational Libraries for the Identification of Bioactive Peptides,” Peptide Research (1992) 5:351-358). Houghten synthesized a peptide combinatorial library (SPCL) composed of some 34×106 hexapeptides and screened it to identify antigenic determinants that are recognized by a monoclonal antibody. Furka et al., “General Method for Rapid Synthesis of Multicomponent Peptide Mixtures,” Int. J. Peptide Protein Res. (1991) 37:487-493, discusses a method of synthesizing multicomponent peptide mixtures. Furka proposed pooling as a general method for the rapid synthesis of multicomponent peptide mixtures and illustrated its application by synthesizing a mixture of 27 tetrapeptides and 180 pentapeptides. Lam et al., “A new type of synthetic peptide library for identifying ligand-binding activity,” Nature (1991) 354:82-84 used pooling to generate a pentapeptide bead library that was screened for binding to a monoclonal antibody. Blake et al. “Evaluation of Peptide Libraries: An Interactive Strategy To Analyze the Reactivity of Peptide Mixtures With Antibodies,” Bioconjugate Chem. (1992) 3:510-513 discusses the screening of presumed mixtures of 50,625 tetrapeptides and 16,777,216 hexpeptides to select epitopes recognized by specific antibodies.
Lam's synthetic peptide library consists of a large number of beads, each bead containing peptide molecules of one kind. Beads that bind a target (e.g., an antibody or strepavidin) are rendered colored or fluorescent. Lam reports that several million beads distributed in 10-15 petri dishes can be screened with a low-power dissecting microscope in an afternoon. Positive beads are washed with 8M guanidine hydrochloride to remove the target protein and then sequenced. The 100-200 μm diameter beads contain 50-200 pmol of peptide, putatively well above their 5 pmol sensitivity limit. Three pentapeptide beads were sequenced daily. The essence of Lam's method is that the identity of positive beads is established by direct sequencing.
Houghton et al. use a different approach to identify peptide sequences that are recognized by an antibody. Using the nomenclature described herein, Houghton et al. screened an X6X5X4pX3pX2pX1p library and found that the mixture DVX4pX3pX2pX1p had the greatest potency in their inhibition assay. Houghton et al. then synthesized a DVX4X3pX2pX1p library and identified the most potent amino acid in the third position. After three more iterations, they found that DVPYDA (SEQ ID NO: 1) binds to the antibody with a Kd of 30 nM. The essence of Houghten's method is recursive retrosynthesis, in which the number of pooled positions decreases by one each iteration.
Blake et al. used a “bogus coin strategy” to guide them to a preferred amino acid sequence. In this strategy a basis set of monomers (15 amino acids) is first divided into three groups. Blake et al. chose A, L, V, F, Y (subgroup α), G, S, P, D, E (subgroup β), and K, R, H, N, Q (subgroup γ). By adjusting the “weighting” of the subgroups at each position in the polymer sequence, and then testing the activity of the weighted polymer against an unweighted polymer, one subgroup was selected for each monomer position in the sequence. In an experiment conducted by Blake et al., a complete collection of tetramers X1pX2pX3pX4p was reduced to α1α2γ3α4 by four inhibition experiments. Then the subgroups α and γ were each further subdivided into three groups of amino acid which were used to synthesize four more collections of weighted polymers. Inhibition studies with each of these collections suggested an epitope (F or Y)1 (A or L)2 (K or R)3 (F or Y)4. One more iteration gave the desired epitope FLRF(SEQ ID NO: 2).
While meeting with some success, prior methods have also met with certain limitations. For example, it is sometimes desirable to avoid the use of the equipment necessary to conduct light directed techniques. Also, some prior methods have not produced the desired amount of diversity as efficiently as would be desired.
From the above, it is seen that an improved method and apparatus for synthesizing a diverse collection of chemical sequences is desired.